The goal of the proposed research is to generate and extensively characterize high passage and immortalized adult human microglia cell lines. High yield and well characterized adult human microglia cultures from each case will be necessary for mechanistic in vitro studies. Availability of large numbers of cells would also allow initial testing of drugs that can regulate microglial functions and permit isolation of large amounts of RNA and/or protein for transcriptomic or proteomic studies. Microglia have diverse functions in the healthy and diseased brain. A great deal of what has been learned regarding microglia biology is based on in vitro studies the overwhelming majority of which used cells isolated from the rodent brain. However, higher anatomical and functional complexity of the human brain and species differences in microglia response and function make imperative the use of human microglia to ascertain that the results obtained are applicable to man. Furthermore, investigation of microglia functions in the adult brain, in which many inflammatory and anti- inflammatory microglia responses occur, requires use of human microglia from adult brains. Microglia cultured from embryonic human brain show substantial proliferative capacity. However, while methods for isolation of microglia from adult postmortem human brains exist, they allow only use of a limited quantity of microglia isolated and cultured from each case due to low levels of proliferation. In preliminary attempts, we have developed a method that allows culturing microglia from adult postmortem human brains to relatively high passage. Limited preliminary testing indicated that human microglia may maintain their phenotype in high passage cultures. The proposed work will attempt to generate microglia cultures of higher passage and to extensively characterize microglia phenotypes and response to different stimuli at different passages. We will also generate and extensively characterize immortalized human microglia cell lines using a well-established protocol that utilizes human telomerase reverse transcriptase. The specific aims of the proposed work will test the following hypotheses: Aim 1 - Microglia cultured from normal adult human brains of various ages and from brains of individuals with neurodegenerative disorders will proliferate to high passage in vitro. Aim 2 - High passage human microglia will maintain their phenotype, including response to various stimulants. Aim 3 - Human microglia will be successfully immortalized, and immortalized human microglia will maintain their phenotype and response to various stimulants. Successful generation and characterization of high passage and immortalized human microglia cultures will provide the scientific community with new and reliable tools for in vitro mechanistic studies of human microglia function in health from childhood to old age, and in disease, including our own studies of microglia function in Alzheimer's disease.